UBE2C promotes leptomeningeal dissemination and is a therapeutic target in brain metastatic disease

Abstract Background Despite current improvements in systemic cancer treatment, brain metastases (BM) remain incurable, and there is an unmet clinical need for effective targeted therapies. Methods Here, we sought common molecular events in brain metastatic disease. RNA sequencing of thirty human BM identified the upregulation of UBE2C, a gene that ensures the correct transition from metaphase to anaphase, across different primary tumor origins. Results Tissue microarray analysis of an independent BM patient cohort revealed that high expression of UBE2C was associated with decreased survival. UBE2C-driven orthotopic mouse models developed extensive leptomeningeal dissemination, likely due to increased migration and invasion. Early cancer treatment with dactolisib (dual PI3K/mTOR inhibitor) prevented the development of UBE2C-induced leptomeningeal metastases. Conclusions Our findings reveal UBE2C as a key player in the development of metastatic brain disease and highlight PI3K/mTOR inhibition as a promising anticancer therapy to prevent late-stage metastatic brain cancer.

Dissemination of cancer cells to the brain is a frequent and late-stage complication of many systemic cancers. Patients with brain metastatic disease have a poor prognosis, with a median survival between 3 and 11 months. 1,2 Lung cancer (40%-60%), breast cancer (15%-30%), and melanoma (5%-15%) are the most common primary malignancies that disseminate to the brain. [2][3][4][5] Autopsy studies showed that around 20%-26% of all cancer patients develop brain metastases (BM) but it is thought that the incidence may be higher and increasing. 3,5,6 Advanced methods of BM diagnosis and more effective treatments for extracranial disease led to an improvement in patients' survival, thus contributing to an increase in BM incidence. The standard of care treatment for BM includes surgical resection, wholebrain radiotherapy (WBRT), and stereotactic radiosurgery (SRS). 7 Nonetheless, brain metastatic disease remains incurable. 3 Furthermore, the most aggressive form of this disease is characterized by coating of the leptomeninges by cancer cells (leptomeningeal dissemination), and poor response to intensive chemotherapeutic regimens, including intrathecal therapy. 8 Currently, molecular-targeted therapies in use for primary tumors lack good therapeutic responses in metastatic tumors located in the central nervous system (CNS). This might be explained either by the limited crossing of drugs through the blood-brain barrier (BBB) 9 or by genetic differences between primary tumors and BM. 10 Understanding the molecular mechanisms underlying the dissemination of cancer cells into the brain will open the possibility of identifying targets to be used in the development of novel therapies. Previous attempts to study the mechanisms of cancer dissemination to the brain focused mainly on analyzing genes differentially expressed between the most common primary tumors (lung and breast cancer) and their BM. The metastatic compartment often exhibits a distinct genetic profile from the primary tumor. 10 Specific molecular targets have been found to play a role in the metastatic process to the brain, namely

Importance of the Study
Brain metastases (BM) are a late-stage complication in cancer patients and remain an incurable disease. Using RNA sequencing and tissue microarrays in surgically resected BM from cancer patients with diverse primary cancers, we have identified UBE2C as a molecular marker of prognosis in patients with brain metastatic disease. High expression of UBE2C was associated with patients' worse survival. We have shown that UBE2C increases migration and invasion and induces leptomeningeal dissemination in vivo. Importantly, early treatment of UBE2C-driven mouse and patientderived xenografts with the dual PI3K/mTOR inhibitor dactolisib, prevented leptomeningeal dissemination, via downregulation of UBE2C.
The identification of UBE2C as a molecular marker of prognosis in brain metastatic disease across multiple cancer types and the ability to prevent leptomeningeal dissemination by UBE2C indirect targeting, open a new avenue for the designing of clinical trials toward the prevention of late-stage metastatic brain cancer.
Paisana et al.: UBE2C promotes brain metastatic disease HER2, FOXC1, LEF1, and HOXB9 in lung cancer, COX2, HBEGF, and ST6GALNAC5 in breast cancer, and plasminogen activator (PA) inhibitory serpins in both these tumors. [11][12][13] We hypothesized that brain metastatic tumors from diverse histological types share common genetic events which promote cancer cell dissemination and colonization of the brain, and these molecular alterations can be used as therapeutic targets. We performed RNA sequencing in a discovery cohort of thirty BM samples from patients diagnosed with various primary tumors and identified UBE2C as being differentially expressed. UBE2C is a ubiquitin-conjugating enzyme that functions together with anaphase-promoting complex/cyclosome (APC/C) involved in the cell cycle, ensuring a correct transition from metaphase to anaphase. 14,15 UBE2C is present in the 20q13.1 locus, a region known to be amplified in a variety of malignancies, including gastro-esophageal carcinomas. 16 High expression of UBE2C has been found in tumor tissues from different histological types, and it was associated with a worse prognosis in primary cancers. 16,17 We analyzed an independent cohort of 89 BM from different primary origins and observed that patients with higher expression of UBE2C had a significantly worse prognosis. In orthotopic mouse models of BM, UBE2C promoted leptomeningeal dissemination and decreased survival, possibly due to an increase in cancer cell migration and invasion. Importantly, early treatment with a PI3K/ mTOR inhibitor (dactolisib) prevented the UBE2C-induced leptomeningeal dissemination in these models, revealing a promising therapeutic avenue in advanced metastatic cancer prevention.

Key Resources
A full description of Materials and methods can be found in Supplementary Materials document.

Patients' Samples
BM samples were collected in accordance with the Ethics board from Hospital de Santa Maria (Refª. Nº 367/18 and Refª. Nº 346/20) and a written informed consent was obtained from all patients, prior to study participation.

RNA Sequencing Analysis
The BM samples from diverse primary origins included in the study reflect the series of patients submitted to brain surgery at the Department of Neurosurgery (HSM-CHULN). Total RNA isolated from BM was processed using the TruSeq RNA Sample Preparation v2 kit (lowthroughput protocol; Illumina, San Diego, CA, USA) to prepare the barcoded libraries. Libraries were validated and quantified using either DNA 1000 or high-sensitivity chips on a Bioanalyzer (Agilent, Santa Clara, CA, USA). 7.5 pM denatured libraries were input into cBot (Illumina), followed by deep sequencing using HiSeq 2500 (Illumina) for 101 cycles, with an additional seven cycles for index reading. For normal tissue samples, we downloaded the call sets from the ENCODE portal (https://www. encodeproject.org/) for tissues matching the tissue of origin of the BM.
Fastq files were imported into Partek Flow (Partek Incorporated, St. Louis, MO, USA). Quality analysis and quality control were performed on all reads to assess read quality and to determine the amount of trimming required (both ends: 13 bases 5ʹ and 1 base 3ʹ). Trimmed reads were aligned against the hg38 genome using the STAR v2.4.1d aligner. Unaligned reads were further processed using Bowtie 2 v2.2.5 aligner. Finally, aligned reads were combined before quantifying the expression against the ENSEMBL (release 84) database using the Partek Expectation-Maximization algorithm. Partek Flow default settings were used in all analyses. Files were then processed using Partek Genomic Suite (Partek Incorporated, St. Louis, MO, USA). Genes were filtered for expression values ≤ 1 and 3 or more missing values, the remaining genes were then log2 transformed.

Microarray Datasets used in the Bioinformatic Analysis
Microarray datasets were also used in the analysis of RNA sequencing. GSE2109 and GSE7307 datasets were downloaded from GEO DataSets (https://www.ncbi.nlm.nih.gov/ gds) and processed using Partek Genomic Suite. Files were imported into Partek Genomic Suites and normalized using the RMA method.

Cell Culture
Human cell lines MDA-MB-231 (referred as MDA), A549 and HCT116 were maintained in the appropriate media. We induced brain tropism in these cell lines as previously described. 12 All cell lines were genetically modified to overexpress UBE2C and MDA, additionally, for the KD of UBE2C.
MET-CF78 cells were derived from a patient with lung cancer BM (patient-derived cell culture), established in our laboratory as previously described. 18 Paisana et al.: UBE2C promotes brain metastatic disease

Cell Modulation
To achieve stable overexpression, the lentiviral vector was used for gene delivery. The plasmid for human UBE2C overexpression was made by subcloning the PCRamplified UBE2C (IDT) fragment into the EcoRI and Mscl (NEB) site of LeGO-iV2.

Immunoblotting
Whole-cell lysates were prepared as previously described 19 and the immunoblotting was performed according to standard procedures. Two independent experiments were performed.

Cell Proliferation
CellTiter 96 Aqueous One Solution Reagent (MTS) was used as defined by the manufacturer's protocol, for the time points of 0, 24, 48, 72, and 96 hours. Three independent experiments, with 3 technical replicates.

Colony Formation Assay
MDA or A549 were seeded onto 6 well plates, with 100 cells/well, and incubated at 37°C, 5% CO 2 . Cells were allowed to grow for two weeks. Pictures were analyzed using ColonyArea plugin for ImageJ. 20 Migration Seeding 2 × 10 4 cells in CIM-plate 16 and the impedance signals were recorded using the xCELLigence, 21 for 72 hours.

In Vivo Orthotopic Xenografts
In accordance with Directive 2010/63/EU (transposed to Portuguese legislation through Decreto-Lei No. 113/2013, of August 7th), all animal procedures were approved by the institutional animal welfare body (ORBEA-iMM). NSG mice were acquired from Charles River Laboratories or in the NSG colony established in-house. Animals were subjected to procedures between the ages of 11 and 22 weeks old. Cancer cells were injected intracranially. MDA models were established by injecting 50,000 cells and 100,000 in MET-CF78 model. Histopathologic analysis was performed in CNS samples.

Mice In Vivo Imaging
Mice were injected with XenoLight D-Luciferin, Potassium Salt and imaged after 10 minutes in IVIS Lumina System, 5 minutes exposure.

Drug Screening
A series of six-nine dilution steps of each inhibitor between 32.5 and 25,000 nM was tested. About 30 µl of cell suspension were seeded into each well of the drug library. After 72 hours of incubation at 37°C and 5% CO 2 , plates were analyzed using CellTiter-Glo reagent and read in the Spark MultiMode Plate reader.

Drug Testing in Orthotopic BM Xenografts
NSG mice injected intracranially with MDA cancer cell line (5 × 10 4 cells/mouse) or MET-CF78 (1 × 10 5 cells/mouse) were treated with dual ATP-competitive PI3K and mTOR inhibitor dactolisib (30 mg/kg, based on the literature 22 ), via oral gavage, daily from day 4 to day 14, for MDA, or from day 7 to day 18, with a wash-out period of 2 days after 5 days of treatment. Animals were randomized into treatment (dactolisib) and control (vehicle: N-Methyl-2-Pyrrolidone (NMP) and Polyethylene glycol 300 (PEG300), 10/90, v/v) groups. By the end of the treatment (day 15 for MDA cells, or 18 for MET-CF78 cells), CNS and organs (lungs, liver, spleen, and kidneys) were collected for histopathological analysis.

UBE2C IHC in Orthotopic BM Xenografts Samples
In mice injected with MDA and treated with dactolisib (or vehicle), UBE2C downregulation was analyzed by an Paisana et al.: UBE2C promotes brain metastatic disease in-house developed macro, using the ImageJ/Fiji macro to quantify the percentage of high UBE2C-staining in the tumor tissue (available at https://github.com/ClaraBarreto/ UBE2C).

Statistical Analysis
For statistical differences using multiple testing, as in the RNA sequencing analysis, a Bonferroni adjusted p-value was used. Other statistical differences were determined using t-test (parametric) or Mann-Whitney tests (nonparametric) on GraphPad Prism v6.0 (GraphPad, California, USA, GraphPad Prism, RRID:SCR_002798), as stated in figure legends Differences were considered statistically significant for p ≤ 0.05.

UBE2C is Upregulated in Human BM
A discovery cohort from the neurosurgery department at Hospital de Santa Maria from Centro Hospitalar Universitário Lisboa Norte (HSM-CHULN) comprising thirty human BM from patients diagnosed with different primary tumor origins were analyzed using RNA sequencing. In this cohort, lung cancer was the most frequent primary tumor, followed by breast, uterus, and colon cancer ( Figure 1A). Tumor samples were collected consecutively, and therefore, the representation of each primary tumor origin reflects the frequency in the neurosurgical series. The transcriptomic analysis included publicly available RNA sequencing datasets of normal tissues ( Figure  1B) and microarray datasets of normal tissue and primary tumors (Supplementary Figure S1A), matching the BM cohort histological types. Genes upregulated exclusively in BM samples were identified when compared with normal tissue and primary tumors (n = 4514 genes, Figure 1C). From this list, we selected the top 20 upregulated genes in BM based on p-value ≤ 0.05 and positive or negative fold change ≥ 1.7 ( Figure 1D and Supplementary Figure S1B). The expression of these genes was then checked for directionality in all tumor types and the most promising genes were further investigated regarding their clinical relevance. Among the top 5 candidate genes (UBE2C, ASF1B, FOXM1, HJURP, and KIF18B), UBE2C was the most frequently associated with poor prognosis in publicly available clinical datasets from diverse cancer types, including metastatic melanoma ( Figure 1E). UBE2C was found to be highly expressed in BM samples from diverse primary tumor origins when compared to normal tissues ( Figure 1F).

Patients with High Expression of UBE2C in BM have a Poorer Prognosis
We validated the clinical relevance of UBE2C by immunohistochemistry (IHC) in tissue microarrays (TMAs), using an independent cohort of 89 patients with BM from different primary tumor origins (Figure 2A-B). Samples from patients with primary brain tumors (glioblastoma) were used as control. Patients had a median age of 62 years (28-90 years) (Supplementary Figure S2A), with a similar frequency of male and female patients (52% and 48%, respectively) (Supplementary Figure S2B). The median overall survival of these patients since the diagnosis of BM was 8 months (Supplementary Figure S2C), with different survival rates depending on the primary tumor origin (Supplementary Figure S2D-E). No differences were found in the survival of BM patients when comparing patients with high vs low expression levels (intensity of staining) of ASF1B (Supplementary Figure  S2F) or FoxM1(Supplementary Figure S2G). In contrast, high expression of UBE2C (higher intensity of staining) was associated with a worse prognosis (median survival of 7 months), while patients with low UBE2C expression exhibited a median survival of 12 months (p-value = 0.04) ( Figure 2C). This finding was not due to a higher proliferative index in tumor cells ( Figure 2D-E), nor dependent on the primary tumor type (Supplementary Figure S2H-L). Using a scoring system combining intensity and frequency of UBE2C staining ( Figure 2F), we have observed that most BM samples (55%) have high levels of UBE2C (Score III and IV), in contrast with glioblastomas in which 70% of the samples have low expression of this protein ( Figure 2G). This suggests that high UBE2C expression is a feature of brain metastatic tumors, but not of primary brain tumors, such as glioblastoma.

UBE2C Promotes Cancer Cell Migration and Invasion In Vitro
To validate and characterize the role of UBE2C in the context of brain metastatic disease we used two cancer cell lines, MDA (breast cancer) and A549 (lung cancer). Both cell lines were engineered to stably overexpress UBE2C ( Figure 3A-B). To study the effect of UBE2C in cancer cell migration, we performed real-time cell analyses using the xCELLigence system ( Figure 3C). The migration rate of MDA and A549 cells was significantly increased in UBE2C overexpressing cells ( Figure 3D-E). This effect was not due to an increase in proliferation either in short-term (Supplementary Figure S3A

UBE2C Drives Leptomeningeal Dissemination and Associates with Worse Survival In Vivo
To assess the in vivo role of UBE2C in BM, we performed intracranial injections of MDA cells in NSG mice ( Figure 4A). Animals injected with UBE2C-overexpressing cells showed decreased survival ( Figure 4B, p-value = 0.05), a phenotype associated with more aggressive disease, characterized by leptomeningeal dissemination. Although the levels of leptomeningeal dissemination in the brain were similarly high in both experimental groups (Supplementary Figure  S4A), we observed a significant increase in spinal cord dissemination among animals with UBE2C-overexpressing cells, both in vivo ( Figure 4C-D) and ex vivo ( Figure 4E-G). We also established an MDA cell line with UBE2C knockdown (KD) using the CRISPR system (Supplementary Figure S4B). There were no differences in survival (Supplementary Figure S4C) or dissemination to the brain meninges (Supplementary Figure S4D) in orthotopic models transplanted with UBE2C KD or control cells. However, we found evidence of decreased dissemination of cancer cells with UBE2C silencing to the spinal cord meninges (Supplementary Figure S4E). We also observed that lung cancer sublines with tropism to the leptomeninges have increased levels of UBE2C in comparison with their parental counterparts (Supplementary Figure S4F-G). Thus, UBE2C drives a more aggressive disease phenotype with leptomeningeal dissemination and decreased survival in orthotopic models of BM.

Targeting the PI3K/mTOR Pathway In Vitro Decreases Cancer Cell Proliferation and Downregulates UBE2C
Due to the lack of effective treatment options for patients with BM and the inexistence of specific therapies targeting UBE2C, we performed a high-throughput drug screening to identify compounds with efficacy in treating UBE2Cdriven BM. We used a chemical library of 650 compounds, FDA approved or in phase 3 or 4 clinical trials, to test cell lines with overexpression and knock-down of UBE2C (Supplementary Figure S5A). In this drug screening, we have selected two candidate compounds that targeted cells expressing high levels of UBE2C when compared to their counterparts, for further in vitro validation: dactolisib (PI3K/mTOR inhibitor) and Genz644282 (Topoisomerase I inhibitor) (Supplementary Figure S5B-C). Both compounds proved to be highly effective inhibiting cancer cell proliferation ( Figure 5A-B and S5D-E). Single-cell RNA sequencing data analysis of BM from patients with different primary tumors 23 showed that metastatic tumor cells with high UBE2C expression levels (Supplementary Figure S5F) also presented high levels of MTOR1 (Supplementary Figure S5G), with a positive correlation between the mRNA levels of both genes (Supplementary Figure S5H). In addition, the PI3K/mTOR pathway has been implicated in brain metastatic cancer. 10 Therefore, we decided to further evaluate the efficacy of inhibition of PI3K/mTOR signaling and examine its interplay with UBE2C. Dactolisib effectively inhibited the PI3K/mTOR pathway in breast and lung cancer cell lines in vitro, through the downregulation of pAkt and pS6, respectively ( Figure 5C-D). Furthermore, we have also used a patient-derived culture (PDC), MET-CF78, isolated from a BM-derived from a lung cancer patient 18  Paisana et al.: UBE2C promotes brain metastatic disease constitutive expression of UBE2C ( Figure 5E). We observed inhibition of MET-CF78 proliferation by dactolisib in a dosedependent manner ( Figure 5F). Interestingly, the targeting of PI3K/mTOR signaling pathway by dactolisib decreased the UBE2C levels in all cancer cell lines ( Figure 5G), regardless of their different levels of PI3K activation (Supplementary Figure S5I).

Dactolisib Prevents Leptomeningeal Dissemination In Vivo
We then asked whether the aggressive metastatic phenotype induced by UBE2C in vivo could be prevented by early treatment with dactolisib, a compound known to cross the BBB. 24 A preclinical therapeutic protocol was designed to early treat UBE2C-driven orthotopic mouse models of BM with dactolisib by oral gavage. Treatment was initiated on day 4 post-injection, and it was given daily for 10 days, with a wash-out period of two days ( Figure 6A). There were no differences in weight between dactolisib-treated and untreated animals (Supplementary Figure S6A). Strikingly, although dactolisib was not able to significantly reduce brain tumor size (Supplementary Figure S6B), it was effective in preventing leptomeningeal dissemination both in the brain and spine ( Figure 6C-F), reversing the aggressive phenotype induced by UBE2C-overexpressing cancer cells. Dactolisib-treated brain tumors exhibited a significant decrease in the number of cancer cells expressing high levels of UBE2C ( Figure 6G-H).
In the patient-derived xenograft of the lung cancer BM (MET-CF78), early preclinical treatment with oral dactolisib ( Figure 6I) had no differences in spine dissemination (Supplementary Figure S6C) and there was no impact of the treatment in the weight of the animals (Supplementary Figure S6D). However, dactolisib significantly reduced both brain tumor size ( Figure 6J-K) and leptomeningeal dissemination to the brain ( Figure 6L-M).
To validate if the effect of dactolisib in vivo was, at least in part, UBE2C-dependent, we developed orthotopic mouse models using MDA cells with KD of UBE2C (n = 7 mice/ group). There were no differences in the weight of treated animals compared to the untreated (Supplementary Figure  S6E). In UBE2C-depleted tumors, dactolisib did not significantly impact brain tumor size (Supplementary Figure S6F

Discussion
Despite significant therapeutic advances in the treatment of primary cancers, BM remain a major clinical hurdle. We began addressing this challenge by interrogating BM from diverse primary cancers using RNA sequencing coupled with functional approaches, in both human and mouse models. Focusing on BM from diverse primary tumors, we were able to identify common genetic events leading to cancer cell dissemination and/or colonization into the brain. Simultaneously, these molecular targets constitute novel targets for therapy.
We identified UBE2C as a differentially expressed gene in human BM and showed that high levels of UBE2C are associated with shorter survival in cancer patients with brain metastatic disease. UBE2C was first described by Okamoto as an oncogene, overexpressed in primary tumors and cancer cell lines, when compared to normal tissues. 17 It has also been shown that UBE2C correlates with higher tumor grades. In lung cancer patients, UBE2C was associated with poorer survival 25 and with tumor progression, as a consequence of autophagy inhibition and increased tumor invasiveness. 26 In breast cancer, UBE2C was identified as a prognostic marker 27 and associated with cancer grade progression 28 and drug resistance. 29 Similar observations were made in colon, ovarian, and bladder cancers, among other types of tumors. [30][31][32][33] Interestingly, UBE2C was associated with BM in a recent publication, where single-cell RNA sequencing of BM from multiple cancers led to the identification of two subgroups, being UBE2C one of the signature genes in the proliferative group. 23 UBE2C has been described as a promoter of migration and invasion of tumor cells in endometrial cancer models, inducing epithelial to mesenchymal transition (EMT), via downregulation of p53 levels. 34 Similar effects were also observed in gastric 35 and hepatocellular cancers. 36 We observed that orthotopic mouse models injected with cancer cells overexpressing UBE2C developed a more aggressive disease phenotype, with leptomeningeal dissemination in the brain and spinal cord. This phenomenon may be explained by the increase in cancer cell migration and invasion induced by overexpression of this gene, as observed in vitro. One possible explanation for the increased colonization of the leptomeninges, particularly in the spinal cord, in a poor microenvironment such as the cerebrospinal fluid (CSF), is that cancer cells with high levels of UBE2C have higher glycolytic activity, further promoting their migration and invasion abilities even in such environmental conditions. 37,38 In fact, a metabolic diagnostic tool based on nuclear magnetic resonance (NMR) analysis of patients with leptomeningeal carcinomatosis showed increased lactate levels, a product of glycolysis. 39 In cancer patients, leptomeningeal dissemination can occur in approximately 5%-15% of the cases, 8 having a dismal prognosis with a median survival between 2 and 4 months. 8,[40][41][42] Very little is known about the molecular biology of this advanced stage of metastatic cancer, making it exceedingly difficult to treat. Aggressive therapies including intrathecal chemotherapy and whole-brain radiation have failed to alter the natural history of leptomeningeal dissemination. Recent phase 2 studies have reported the clinical benefit of patients treated with EGRF inhibitors and immunotherapy, 43,44 although the duration of responses in leptomeningeal disease was limited in time.
Since late-stage CNS dissemination seems very difficult to target, the best chance for improved survival in these patients would be to prevent cancer cells from disseminating into the leptomeninges. Our observations revealed that early treatment with oral dactolisib (a dual PI3K/mTOR inhibitor) prevented the development of leptomeningeal dissemination in orthotopic mouse models driven by UBE2C. This effect of dactolisib might be UBE2C-dependent because, in UBE2C-depleted tumors, treatment did not reduce brain tumor size or CNS dissemination.